The immunoglobulin G fragment crystallizable region (IgG-Fc) can be used as a scaffold to attach drugs and polymers. However, when chemical attachment to their amine groups is attempted, a heterogeneous mixture of attachments results, which is undesirable for therapeutic or diagnostic use. (Panowski et al., mAbs, 6:34-45 (2014).
IgG-Fc domains are commonly used in the art to create IgG-Fc fusions with a variety of polypeptides (Czajkowsky et al., EMBO Mol Med, 4:1015-1028 (2012)). In some cases, the polypeptide to be fused may be devoid of lysine residues. In such cases, only the primary amine group of the polypeptide at the N-terminus of the fusion polypeptide represents a unique attachment site. Alternatively, the primary amine group at the N-terminus may be eliminated if the N-terminal amino acid is glutamine or glutamate and is subsequently converted to pyroglutamate. (Lui et al., J. Biol. Chem, 286:11211-11217 (2011)). As early as 2007, over two dozen monoclonal antibodies and Fc fusion proteins had been approved as therapeutic products, including blockbuster products such as Rituxan® (Genentech; Biogen-Idec), Remicade® (Centocor), Herceptin® (Genentech), Avastin® (Genentech), Bexxar® (Glaxo SmithKline), Tysabri® (Biogen-Idec), Humira® (Abbott), Enbrel® (Amgen); and Campath® (Takeda-Millennium).
The Fc portion of human IgG1 contains 20 native lysine residues, resulting in a total of 40 lysines present in the functional dimeric Fc. Chemical conjugation, using amine reactive chemistries results in heterogenous attachments at these naturally occurring lysines. Moreover, several of the native lysines near and within the CH2-CH3 interface may contribute to the binding interaction with protein A. For example, lysine 338 (by Kabat EU numbering) that is conserved in several species of IgG has been identified as a critical residue for the binding of IgG to Staphylococcal protein A (hereafter referred to as “protein A”) (Deisenhofer (1981) Biochemistry, 20:2361-2370; Nagaoka and Akaike (2003), Protein Engineering (2003) 16:243-5). The disclosures of the above documents are hereby incorporated by reference.
U.S. Pat. No. 8,889,629 discloses fusion molecules comprising two or more PSGL-1 domains fused to an IgG-Fc domain.